Recombinant Danio rerio E3 ubiquitin-protein ligase arih1 (arih1), partial

What is ARIH1 and what are its primary cellular functions?

ARIH1 (Ariadne RBR E3 Ubiquitin Protein Ligase 1) functions as an E3 ubiquitin ligase that catalyzes the transfer of ubiquitin to target proteins, marking them for degradation or modifying their function. It plays critical roles in multiple biological processes including antiviral immunity and neurological function. In immunity, ARIH1 catalyzes the mono-ISGylation of cyclic GMP-AMP synthase (cGAS), a key sensor in antiviral immunity, promoting its oligomerization and activation . In neurological contexts, ARIH1 regulates G protein-gated inwardly rectifying potassium channel 2 (GIRK2) degradation in hippocampal neurons, which impacts learning and memory functions . These diverse roles highlight ARIH1's importance in cellular homeostasis across different physiological systems.

How is ARIH1 conserved across species from zebrafish to mammals?

ARIH1 demonstrates significant evolutionary conservation from zebrafish (Danio rerio) to mammals, indicating its fundamental biological importance. The availability of recombinant Danio rerio E3 ubiquitin-protein ligase arih1 for research purposes reflects this conservation . Research shows that ARIH1 homologs in C. elegans have been implicated in fertility and oogenesis during development . The functional conservation is evident in the ubiquitination processes across different species, though specific targets may vary between organisms. This conservation allows researchers to use zebrafish models to study ARIH1 functions that may be relevant to human biology and disease mechanisms.

What purification methods ensure optimal activity of recombinant ARIH1?

According to product specifications, recombinant Danio rerio ARIH1 is typically produced with a purity of >85% as verified by SDS-PAGE . To ensure optimal activity when working with this protein, researchers should consider several purification validation steps. First, verify protein purity through SDS-PAGE analysis, which allows visualization of the target protein band and potential contaminants. Second, conduct Western blotting with ARIH1-specific antibodies to confirm identity. Third, assess enzymatic activity through in vitro ubiquitination assays using known ARIH1 substrates. Finally, the source of recombinant protein (E. coli expression systems in this case) may affect post-translational modifications and folding, potentially impacting enzymatic activity compared to native ARIH1 .

What are the optimal storage and handling conditions for recombinant ARIH1?

Optimal storage and handling of recombinant Danio rerio ARIH1 requires careful attention to temperature, formulation, and freeze-thaw cycles. According to product specifications, liquid formulations generally have a shelf life of 6 months when stored at -20°C/-80°C, while lyophilized formulations extend this to 12 months at the same temperatures . Repeated freezing and thawing is explicitly not recommended, suggesting researchers should aliquot the protein upon receipt to minimize freeze-thaw cycles . For short-term use, working aliquots can be stored at 4°C for up to one week .

How should recombinant ARIH1 be reconstituted for experimental applications?

For optimal reconstitution of recombinant Danio rerio ARIH1, the product specifications provide a detailed protocol. First, briefly centrifuge the vial prior to opening to ensure all content is at the bottom . Next, reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For long-term storage preparation, it is recommended to add glycerol to a final concentration between 5-50%, with 50% being the default recommendation, which helps prevent damage from freeze-thaw cycles and maintains protein stability . After reconstitution, the solution should be aliquoted and stored at -20°C/-80°C to prevent degradation from repeated freeze-thaw cycles. This careful reconstitution process is essential for maintaining the structural integrity and enzymatic activity of the protein for subsequent experimental applications.

What experimental considerations are important when using partial versus full-length ARIH1?

When working with partial recombinant Danio rerio ARIH1 as opposed to full-length protein, researchers must consider several important experimental factors. First, determine which functional domains are present in the partial protein to ensure it contains the regions necessary for your specific application . The partial protein described in product specifications may lack certain domains that could influence interaction with substrates or cofactors. Second, validate whether the partial protein retains the enzymatic activity needed for your experiments through appropriate ubiquitination assays. Third, include parallel experiments with full-length ARIH1 when possible to identify any functional differences. Finally, when designing control experiments, consider that partial proteins may exhibit altered stability, solubility, or subcellular localization compared to their full-length counterparts, potentially necessitating adjustments to experimental conditions.

How does ARIH1 mechanistically regulate cGAS-mediated antiviral responses?

ARIH1 plays a sophisticated role in regulating cGAS-mediated antiviral responses through post-translational modifications. Mechanistically, ARIH1 catalyzes the mono-ISGylation of cGAS at the K187 residue, which potentiates its oligomerization and activation in response to cytosolic DNA or HSV-1 infection . This modification enhances cGAS enzymatic activity, leading to increased production of cGAMP, which subsequently activates downstream type I interferon responses. Experimental evidence demonstrates that knockdown or knockout of ARIH1 significantly inhibits HSV-1- or cytoplasmic DNA-induced expression of type I interferons and proinflammatory cytokines .

The physiological importance of this regulatory mechanism is further supported by in vivo studies showing that ARIH1-deficient mice (tamoxifen-treated ER-Cre;Arih1 fl/fl mice and Lyz2-Cre; Arih1 fl/fl mice) are hypersensitive to HSV-1 infection compared to controls . This mechanistic pathway represents a critical regulatory node in innate antiviral immunity, where ARIH1-mediated modifications fine-tune the sensitivity and amplitude of immune responses to pathogenic DNA.

What experimental approaches best demonstrate ARIH1's impact on viral immunity?

To effectively demonstrate ARIH1's impact on viral immunity, researchers have employed multiple complementary experimental approaches. First, genetic manipulation through knockdown or knockout of ARIH1 in cell lines (HEK293, THP-1) has revealed its importance in DNA-sensing pathways . Second, primary immune cell models using bone marrow-derived dendritic cells (BMDCs) from ARIH1-deficient mice have shown impaired responses specifically to HSV-1 or cytoplasmic DNA, but not to RNA virus stimuli (SeV, poly(I:C)) or downstream signaling activators (cGAMP) .

Third, in vivo infection models using conditional knockout mice (tamoxifen-treated ER-Cre;Arih1 fl/fl and Lyz2-Cre;Arih1 fl/fl) have demonstrated increased susceptibility to HSV-1 infection . Fourth, biochemical approaches including ubiquitination/ISGylation assays have identified the specific modification (mono-ISGylation) and target residue (K187) on cGAS . Fifth, mechanistic studies showing cGAS oligomerization in the presence or absence of ARIH1 have connected the molecular modification to functional outcomes. This multi-layered experimental strategy provides robust evidence for ARIH1's role in viral immunity while distinguishing between direct and indirect effects.

How does ARIH1 function in autoimmune disease contexts?

ARIH1 exhibits a dual role in immunity, enhancing antiviral defense while potentially exacerbating autoimmune conditions. Research demonstrates that deletion of ARIH1 in myeloid cells alleviates autoimmune phenotypes and completely rescues the autoimmune lethality caused by TREX1 deficiency . TREX1 is a DNA exonuclease that prevents accumulation of cytosolic DNA, and its deficiency leads to inappropriate activation of cGAS-STING pathways and subsequent autoimmunity.

This finding suggests that ARIH1 inhibition could potentially serve as a therapeutic strategy for autoimmune diseases characterized by aberrant activation of cytosolic DNA-sensing pathways, such as Aicardi-Goutières syndrome and systemic lupus erythematosus. The molecular mechanism involves ARIH1's enhancement of cGAS activation through mono-ISGylation, which becomes pathological in contexts where self-DNA inappropriately triggers immune responses . This highlights the delicate balance between effective antimicrobial defense and prevention of self-reactivity, with ARIH1 functioning as a critical regulatory node that could be therapeutically targeted.

Through what mechanisms does ARIH1 deficiency affect spatial learning and memory?

ARIH1 deficiency impairs spatial learning and memory through disruption of hippocampal function via specific molecular mechanisms. Research using heterozygous ARIH1+/- mice has demonstrated significant deficits in spatial learning and memory in the Morris water maze test, showing longer latency in finding the platform and fewer platform area crossings compared to wild-type controls . These deficits were observed in both male and female ARIH1+/- mice, indicating sex-independent effects .

The molecular mechanism involves upregulation of G protein-gated inwardly rectifying potassium channel 2 (GIRK2) in the dorsal hippocampus due to impaired ubiquitination and subsequent degradation . GIRK channels regulate neuronal excitability, and their inappropriate upregulation appears to disrupt normal hippocampal circuit function required for spatial learning and memory. The causal relationship between ARIH1 deficiency and memory impairment was confirmed by rescue experiments showing that local restoration of ARIH1 expression in the dorsal hippocampus via lentiviral delivery was sufficient to reverse these memory deficits . This research provides a clear mechanistic pathway linking E3 ubiquitin ligase activity to cognitive function through regulation of ion channel expression.

How does ARIH1 regulate GIRK2 expression in hippocampal neurons?

ARIH1 regulates GIRK2 expression in hippocampal neurons through post-translational control via the ubiquitin-proteasome pathway. Experimental evidence demonstrates that ARIH1 deficiency results in upregulation of GIRK2 protein levels in the dorsal hippocampus . Importantly, knockdown of ARIH1 in cells did not alter mRNA expression of GIRK2, indicating that the regulation occurs at the protein level rather than through transcriptional mechanisms .

The direct molecular mechanism involves ARIH1-mediated ubiquitination of GIRK2, as ubiquitination of GIRK2 was dramatically suppressed in ARIH1 knockdown cells . This provides compelling evidence that ARIH1 functions as an E3 ubiquitin ligase for GIRK2, directly mediating its ubiquitination and subsequent degradation. Under normal physiological conditions, ARIH1-mediated ubiquitination maintains appropriate GIRK2 levels, whereas ARIH1 deficiency leads to GIRK2 accumulation due to reduced degradation. This molecular pathway represents a novel mechanism by which ubiquitin ligases contribute to neuronal function and cognitive processes through regulation of ion channel densities.

What behavioral paradigms effectively detect cognitive deficits associated with ARIH1 deficiency?

Multiple behavioral paradigms can effectively detect cognitive deficits resulting from ARIH1 deficiency, with hippocampal-dependent tasks showing particular sensitivity. Research has successfully employed two complementary behavioral tests: the Morris water maze (MWM) test for spatial learning and memory, and the novel object recognition (NOR) test for recognition memory . Both paradigms detected significant deficits in ARIH1+/- mice compared to wild-type controls.

Importantly, other behavioral domains including locomotor activity, anxiety, and depression-like behaviors showed no significant differences between ARIH1+/- and wild-type mice . This pattern of results indicates that cognitive tests focused on hippocampal-dependent learning and memory processes are most sensitive for detecting ARIH1 deficiency effects, providing a valuable experimental framework for future studies of ARIH1 function in cognition.

How can contradictory findings about ARIH1 function in different model systems be reconciled?

Reconciling seemingly contradictory findings about ARIH1 across different model systems requires careful consideration of context-dependent factors. The search results present ARIH1 functions in distinct biological contexts - immune regulation and neuronal function - which appear disparate but likely reflect cell-type specific roles of this E3 ubiquitin ligase .

To address potential contradictions, researchers should consider: (1) Substrate specificity - ARIH1 targets different proteins in different cell types (cGAS in immune cells versus GIRK2 in neurons); (2) Modification type - ARIH1 catalyzes mono-ISGylation of cGAS but ubiquitination of GIRK2, leading to distinct functional outcomes; (3) Expression patterns of ARIH1 and its co-factors across tissues; (4) Developmental timing influences, as suggested by the embryonic lethality of homozygous ARIH1 knockout .

Experimental approaches to resolve these contradictions should include tissue-specific conditional knockouts (as demonstrated in the research), biochemical assays to identify cell-type-specific substrates, and comprehensive analysis of ARIH1 interaction networks in different cellular contexts. This multifaceted approach can transform apparent contradictions into a more nuanced understanding of ARIH1's context-dependent functions.

What control experiments are essential when studying ARIH1's ubiquitination targets?

Third, ubiquitination assays with catalytically inactive ARIH1 mutants are essential to confirm the dependence on ARIH1's enzymatic activity rather than scaffold functions. Fourth, mass spectrometry analysis of ubiquitinated proteins in control versus ARIH1-deficient samples helps identify specific lysine residues modified and ubiquitin chain types (K48, K63, etc.). Fifth, functional rescue experiments provide crucial evidence for physiological relevance, as shown in research where restoration of ARIH1 expression rescued behavioral deficits .

What methodological approaches can determine whether ARIH1 directly ubiquitinates a candidate substrate?

Determining whether ARIH1 directly ubiquitinates a candidate substrate requires a strategic combination of in vitro, cellular, and in vivo approaches. First, in vitro ubiquitination assays using purified recombinant ARIH1, E1 enzyme, E2 enzyme, ubiquitin, ATP, and the purified candidate substrate can demonstrate direct enzymatic activity. Second, site-directed mutagenesis of predicted ubiquitination sites on the substrate followed by functional testing can identify specific lysine residues targeted by ARIH1.

Third, comparing ubiquitination patterns in wild-type versus ARIH1-deficient systems (as seen in the GIRK2 studies) provides evidence for ARIH1-dependent ubiquitination . Fourth, proximity ligation assays can demonstrate close association between ARIH1 and substrates in intact cells. Fifth, reconstitution experiments in ARIH1-deficient systems using wild-type versus catalytically inactive ARIH1 can distinguish direct enzymatic activity from scaffold functions.

Finally, temporal analysis of substrate ubiquitination following ARIH1 activation or inhibition can demonstrate causality. This multilayered experimental approach generates complementary lines of evidence necessary to establish direct substrate relationships, as exemplified by the methodological rigor in the GIRK2 studies showing dramatically suppressed ubiquitination in ARIH1 knockdown cells .

What are the potential therapeutic applications of targeting ARIH1 in disease contexts?

Research findings suggest dual therapeutic opportunities through ARIH1 modulation in distinct disease contexts. In autoimmune conditions characterized by aberrant DNA sensing, ARIH1 inhibition shows therapeutic promise, as deletion of ARIH1 in myeloid cells alleviates autoimmune phenotypes and completely rescues autoimmune lethality caused by TREX1 deficiency . This suggests potential applications in treating diseases like Aicardi-Goutières syndrome, systemic lupus erythematosus, and other interferonopathies.

Conversely, in neurological disorders characterized by cognitive impairment, ARIH1 augmentation might provide benefits. Research demonstrates that restoring ARIH1 expression in the dorsal hippocampus recovers spatial memory function in ARIH1-deficient mice . Additionally, findings that GIRK2 inhibition improves learning and memory in ARIH1-deficient mice suggest that targeting either ARIH1 or its downstream effectors could provide therapeutic benefits .

These diverse applications highlight the context-dependent nature of ARIH1 functions and underscore the importance of tissue-specific approaches to therapeutic development. Future therapeutic strategies may include selective small molecule modulators of ARIH1 activity, cell-type specific gene therapy approaches, or targeting downstream effectors like GIRK2 in neurological applications.

What are promising future research directions for ARIH1 in cross-disciplinary contexts?

Several promising research directions could advance our understanding of ARIH1 across disciplinary boundaries. First, comprehensive identification of the ARIH1 "substrate-ome" across different tissues would provide insights into its diverse cellular functions. The discovery of cGAS and GIRK2 as substrates in immune and neuronal contexts suggests many additional targets remain unidentified .

Second, investigation of ARIH1's role in neurodevelopment deserves attention, given that homozygous knockout is embryonically lethal and heterozygous mice show cognitive deficits . Third, exploring ARIH1's functions at the intersection of immunity and neuroscience could reveal novel neuroimmune interactions, particularly relevant to neuroinflammatory and neurodegenerative conditions. Fourth, structural studies of ARIH1 in complex with different substrates could guide the development of selective modulators for therapeutic applications.

Finally, translational studies examining ARIH1 expression, activity, and mutations in human diseases could establish its relevance as a biomarker or therapeutic target. This cross-disciplinary approach would leverage insights from both immunology and neuroscience to develop a comprehensive understanding of ARIH1's biological functions and therapeutic potential.